Project Summary Acute and chronic injuries resulting from burns, trauma, and diabetes often result in uncloseable open wounds subject to permanent damage, disfigurement, and potentially death. This is an especially challenging problem where such insults span a relatively large area leaving few sites for potential autologous tissue harvest. The development of replacement bulk tissue equivalents is therefore a major interest in the fields of tissue engineering and regenerative medicine. Commercially available products only address the skin. Whether full-thickness or dermal layer-only, these surface skin grafts cannot fulfill the substantial volume needs of reconstructive surgery. When applied to patients, these grafts often fail due to inability to vascularize in these difficult wound beds. A hemodynamically efficient, patent vascular network is the most important factor governing the engraftment and long-term survival of any replacement tissue. Current approaches to incorporate a vascular network in engineered bulk tissues have succeeded only in generating homogeneous capillary plexuses in microscale (<1 cm3) tissue elements. These networks possess limited hemodynamic control, high vascular resistance, and likely will not thrive if they could be scaled up. We have pioneered the use of tissue biofabrication strategies to develop perfusable vascularized tissue equivalents with heterogeneously sized lumens, which mimics the native microvascular architecture. This proposal will test how prescribed macro-scale vascular network geometries control local microvascular angiogenic response and overall tissue perfusion and engraftment. This proposal has three aims. The first aim is to determine how specific local flow patterns within 3D printed vascular channels influence endothelial cell retention and angiogenic sprouting. The second aim tests whether embedded bulk mesenchymal stem cells augments endothelial retention and sprouting in defined hemodynamic environments. The third aim applies the results of the previous aims and tests the efficacy of rationally designed living 3D printed vascularized tissue equivalents in vivo. An innovative rodent anastomosis model is developed to answer these questions. This proposal will establish and validate a new clinically translatable technology for vascular network graft fabrication. The results will also contribute significant new information about the interplays between endothelial and mesenchymal in response to vessel geometries and fluid flows in vitro and in vivo.